Team:Calgary/Notebook/ProtocolManual/Bsubtilis

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<h1>B. subtilis Protocols</h1>
<h1>B. subtilis Protocols</h1>
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<p>
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<b><i>B. subtilis</i> preparation and transformation (Zhang & Zhang, 2010)</b>
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<ol type=”1”>
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<li>Inoculate <i>B. subtilis</i> SCK6 pAX01-comK cells into 3mL LB medium with 1 μg/mL erythromycin and 1 μg/mL lincomycin in a test tube.</li>
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<li>Cultivate cells overnight at 37°C, shaking at 200rpm. </li>
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<li>Dilute culture to A600 at 1.0 in fresh LB containing 1% (w/v) xylose.</li>
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<li>Grow cells for 2 hours at 37°C, shaking at 200rpm.</li>
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<li>Divide into aliquots and store at -80°C with 10% (w/v) glycerol OR proceed with transformation.</li>
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<li>Add 1μL PCR product to 100μL supercompetent cells in a plastic tube.</li>
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<li>Cultivate cells for 1.5 hours at 37°C, shaking at 200rpm.</li>
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<li>Spread on LB plates (with appropriate antibiotic selection).</li>
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<li>Incubate plates at 37°C for 20 hours.</li>
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</ul>
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<br>
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<b><i>B. subtilis</i> preparation and transformation (modified from Zhang & Zhang, 2010)</b>
 +
 
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<ol type=”1”>
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 +
<li>Inoculate <i>B. subtilis</i> SCK6 pAX01-comK cells into 3mL LB medium with 1 μg/mL erythromycin and 1 μg/mL lincomycin in a test tube.</li>
 +
<li>Cultivate cells overnight at 37°C, shaking at 200rpm. </li>
 +
<li>Take initial absorbance reading at A600 and calculate difference to 1.0; dilute to 1.0 with fresh LB using standard curve. </li>
 +
<li>Add 20% xylose for 2% final xylose concentration. </li>
 +
<li>Grow cells for 2 hours at 37°C, shaking at 200rpm. </li>
 +
<li>Divide into aliquots and store at -80°C with 25% (w/v) glycerol OR proceed with transformation. </li>
 +
<li>Calculate volume for 400ng DNA and add to 100μL supercompetent cells in a plastic tube. </li>
 +
<li>Cultivate cells for >2 hours at 37°C without shaking. </li>
 +
<li>Spread on LB plates (with appropriate antibiotic selection). </li>
 +
<li>Incubate plates at 37°C for 20 hours. </li>
 +
 
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</ul>
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<br>
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<b>Making 2x SG Media for sporulation of <i>B. subtilis</i></b>
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<ol type=”1”>
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<li>Measure 16.0g Difco nutrient broth, 2.0g KCl, and 0.5g MgSO4∙7H2O. Add to large bottle and add distilled water for a 1L solution.</li>
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<li>Adjust the pH to 7.0 and autoclate.</li>
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<li>After cooling, add the following sterile solutions: 1mL of 1M Ca(NO3)2, 1mL of 0.1M MnCl2∙4H2O, 1mL of 1mM FeSO4, and 2mL of 50% (w/v) glucose.</li>
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</ul>
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<br>
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<b>Generating and harvesting <i>B. subtilis</i> spores</b>
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<ol type=”1”>
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<li>Inoculate 10-50mL of LB medium with cells from a fresh colony of <i>B. subtilis</i></li>
 +
<li>Grow for 6-8 hours at 37°C with shaking at 200rpm. </li>
 +
<li>Dilute 1/200 into 50-10000 ml 2xSG medium in an appropriate vessel and grow at 37°C with shaking at 200rpm. </li>
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<li>After 2-3 days, over 90% of the population should be spores. Pellet cells at 9000rpm for 20minutes at low temperatures. </li>
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<li>Wash spores with ice-cold water 8-10 times.</li>
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<li>Store at 4°C with weekly changes of water OR at -20°C for long-term storage.</li>
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</ul>
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</p>
</section>
</section>
</html>
</html>

Revision as of 20:13, 10 October 2014

B. subtilis Protocols

B. subtilis preparation and transformation (Zhang & Zhang, 2010)

  1. Inoculate B. subtilis SCK6 pAX01-comK cells into 3mL LB medium with 1 μg/mL erythromycin and 1 μg/mL lincomycin in a test tube.
  2. Cultivate cells overnight at 37°C, shaking at 200rpm.
  3. Dilute culture to A600 at 1.0 in fresh LB containing 1% (w/v) xylose.
  4. Grow cells for 2 hours at 37°C, shaking at 200rpm.
  5. Divide into aliquots and store at -80°C with 10% (w/v) glycerol OR proceed with transformation.
  6. Add 1μL PCR product to 100μL supercompetent cells in a plastic tube.
  7. Cultivate cells for 1.5 hours at 37°C, shaking at 200rpm.
  8. Spread on LB plates (with appropriate antibiotic selection).
  9. Incubate plates at 37°C for 20 hours.

  10. B. subtilis preparation and transformation (modified from Zhang & Zhang, 2010)
    1. Inoculate B. subtilis SCK6 pAX01-comK cells into 3mL LB medium with 1 μg/mL erythromycin and 1 μg/mL lincomycin in a test tube.
    2. Cultivate cells overnight at 37°C, shaking at 200rpm.
    3. Take initial absorbance reading at A600 and calculate difference to 1.0; dilute to 1.0 with fresh LB using standard curve.
    4. Add 20% xylose for 2% final xylose concentration.
    5. Grow cells for 2 hours at 37°C, shaking at 200rpm.
    6. Divide into aliquots and store at -80°C with 25% (w/v) glycerol OR proceed with transformation.
    7. Calculate volume for 400ng DNA and add to 100μL supercompetent cells in a plastic tube.
    8. Cultivate cells for >2 hours at 37°C without shaking.
    9. Spread on LB plates (with appropriate antibiotic selection).
    10. Incubate plates at 37°C for 20 hours.

    11. Making 2x SG Media for sporulation of B. subtilis
      1. Measure 16.0g Difco nutrient broth, 2.0g KCl, and 0.5g MgSO4∙7H2O. Add to large bottle and add distilled water for a 1L solution.
      2. Adjust the pH to 7.0 and autoclate.
      3. After cooling, add the following sterile solutions: 1mL of 1M Ca(NO3)2, 1mL of 0.1M MnCl2∙4H2O, 1mL of 1mM FeSO4, and 2mL of 50% (w/v) glucose.

      4. Generating and harvesting B. subtilis spores
        1. Inoculate 10-50mL of LB medium with cells from a fresh colony of B. subtilis
        2. Grow for 6-8 hours at 37°C with shaking at 200rpm.
        3. Dilute 1/200 into 50-10000 ml 2xSG medium in an appropriate vessel and grow at 37°C with shaking at 200rpm.
        4. After 2-3 days, over 90% of the population should be spores. Pellet cells at 9000rpm for 20minutes at low temperatures.
        5. Wash spores with ice-cold water 8-10 times.
        6. Store at 4°C with weekly changes of water OR at -20°C for long-term storage.